Mesoscale polynucleotide amplification devices

ABSTRACT

Disclosed are devices for amplifying a preselected polynucleotide in a sample by conducting a polynucleotide amplification reaction. The devices are provided with a substrate microfabricated to include a polynucleotide amplification reaction, chamber, having at least one cross-sectional dimension of about 0.1 to 1000 μm. The device also includes at least one port in fluid communication with the reaction chamber, for introducing a sample to the chamber, for venting the chamber when necessary, and, optionally, for removing products or waste material from the device. The reaction chamber may be provided with reagents required for amplification of a preselected polynucleotide. The device also may include means for thermally regulating the contents of the reaction chamber, to amplify a preselected polynucleotide. Preferably, the reaction chamber is fabricated with a high surface to volume ratio, to facilitate thermal regulation.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.08/745,771 (filed Nov. 8, 1996) now U.S. Pat. No. 6,660,517, which is adivision of U.S. application Ser. No. 08/338,728 (filed Nov. 14, 1994),now U.S. Pat. No. 5,587,128, which is a continuation-in-part of U.S.Ser. No. 08/308,1999 (filed Sep. 19, 1994), now U.S. Pat. No. 5,498,392,which is a continuation of U.S. Ser. No. 07/877,662 (filed May 1, 1992),now abandoned. U.S. application Ser. No. 08/338,728 was filedcontemporaneously with commonly-owned U.S. Ser. No. 08/338,369, which isa continuation-in-part of U.S. Ser. Nos. 07/877,702 (filed May 1, 1992),now abandoned, 08/196,021 (filed Feb. 14, 1994), now U.S. Pat. No.5,635,358 and Ser. No. 08/250,100 (filed May 6, 1994), now abandoned.The disclosures of all of the above-mentioned applications areincorporated herein by reference.

BACKGROUND OF THE INVENTION

This invention relates generally to methods and apparatus for conductingamplification and various analyses of polynucleotides. Moreparticularly, the invention relates to the design and construction ofsmall, typically single-use, modules for use in analyses involvingpolynucleotide amplification reactions such as the polymerase chainreaction (PCR).

In recent decades, the art has developed a very large number ofprotocols, test kits, and cartridges for conducting analyses ofbiological samples for various diagnostic and monitoring purposes.Immunoassays, immunometric assays, agglutination assays and analysesbased on polynucleotide amplification assays (such as polymerase chainreaction), or on various ligand-receptor interactions and/ordifferential migration of species in a complex sample, all have beenused to determine the or concentration of various biological compoundsor contaminants, or the presence of particular cell types.

Recently, small, disposable devices have been developed for handlingbiological samples and for conducting certain clinical tests. Shoji etal. reported the use of a miniature blood gas analyzer fabricated on asilicon wafer. Shoji et al., Sensors and Actuators, 15:101-107 (1988).Sato et al. reported a cell fusion technique using micromechanicalsilicon devices. Sato et al., Sensors and Actuators, A21-A23:948-953(1990). Ciba Corning Diagnostics Corp. (USA) has manufactured amicroprocessor-controlled laser photometer for detecting blood clotting.

Micromachining technology, using, e.g., silicon substrates, has enabledthe manufacture of microengineered devices having structural elementswith minimal dimensions ranging from tens of microns (the dimensions ofbiological cells) to nanometers (the dimensions of some biologicalmacromolecules). Angell et al., Scientific American, 248:44-55 (1983).Wise et al., Science, 254:1335-42 (1991); and Kricka et al., J. Int.Fed. Clin. Chem., 6:54-59 (1994). Most experiments involving structuresof this size relate to micromechanics, i.e., mechanical motion and flowproperties. The potential capability of these structures has not beenexploited fully in the life sciences.

Brunette (Exper. Cell Res., 167:203-217 (1986) and 164:11-26 (1986))studied the behavior of fibroblasts and epithelial cells in grooves insilicon, titanium-coated polymers and the like. McCartney et al. (CancerRes., 41:3046-3051 (1981)) examined the behavior of tumor cells ingrooved plastic substrates. LaCelle (Blood Cells, 12:179-189 (1986))studied leukocyte and erythrocyte flow in microcapillaries to gaininsight into microcirculation. Hung and Weissman reported a study offluid dynamics in micromachined channels, but did not produce dataassociated with an analytic device. Hung et al., Med. andBiol-Engineering, 9:237-245 (1971); and Weissman et al., Am. Inst. Chem.Eng. J., 17:25-30 (1971). Columbus et al. utilized a sandwich composedof two orthogonally orientated v-grooved embossed sheets in the controlof capillary flow of biological fluids to discrete ion-selectiveelectrodes in an experimental multi-channel test device. Columbus etal., Clin. Chem., 33:1531-1537 (1987). Masuda et al. and Washizu et al.have reported the use of a fluid flow chamber for the manipulation ofcells (e.g., cell fusion). Masuda et al., Proceedings IEEE/IAS Meeting,pp. 1549-1553 (1987); and Washizu et al., Proceedings IEEE/IAS Meetingpp. 1735-1740 (1988). Silicon substrates have been used to developmicrodevices for pH measurement and biosensors. McConnell et al.,Science, 257:1906-12 (1992); and Erickson et al., Clin. Chem., 39:283-7(1993). However, the potential of using such devices for the analysis ofbiological fluids heretofore has remained largely unexplored.

Methodologies for using polymerase chain reaction (PCR) to amplify asegment of DNA are well established. (See, e.g., Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1989, pp. 14.1-14.35.) A PCR amplification reaction can beperformed on a DNA template using a thermostable DNA polymerase, e.g.,Taq DNA polymerase (Chien et al. J. Bacterial., 127:1550 (1976)),nucleoside triphosphates, and two oligonucleotides with differentsequences, complementary to sequences that lie on opposite strands ofthe template DNA and which flank the segment of DNA that is to beamplified (“primers”). The reaction components are cycled between ahigher temperature (e.g., 94° C.) for dehybridizing (“melting”) doublestranded template, DNA, followed by lower temperatures (e.g., 40-60° C.for annealing of primers and, e.g., 70-75° C. for polymerization). Arepeated reaction cycle between dehybridization, annealing andpolymerization temperatures provides approximately exponentialamplification of the template DNA. For example, up to 1 μg of target DNAup to 2 kb in length can be obtained from 30-35 cycles of amplificationwith only 10⁻⁶ μg of starting DNA. Machines for performing automated PCRchain reactions using a thermal cycler are available (Perkin ElmerCorp.)

Polynucleotide amplification has been applied to the diagnosis ofgenetic disorders (Engelke et al., Proc. Natl. Acad. Sci., 85:544(1988), the detection of nucleic acid sequences of pathogenic organismsin clinical samples (Ou et al., Science, 239:295 (1988)), the geneticidentification of forensic samples, e.g., sperm (Li et al., Nature,335:414 (1988)), the analysis of mutations in activated oncogenes (Farret al., Proc. Natl. Acad. Sci., 85:1629 (1988)) and in many aspects ofmolecular cloning (Oste, BioTechniques, 6:162 (1988)). Polynucleotideamplification assays can be used in a wide range of applications such asthe generation of specific sequences of cloned double-stranded DNA foruse as probes, the generation of probes specific for uncloned genes byselective amplification of particular segments of cDNA, the generationof libraries of cDNA from small amounts of mRNA, the generation of largeamounts of DNA for sequencing, and the analysis of mutations.

A wide variety of devices and systems has been described in the art forconducting polynucleotide amplification reactions using thermal cyclingprocedures. Templeton, Diag. Mol. Path., 1:58-72 (1993); Lizardi et.al., Biotechnology, 6:1197-1202 (1988); Backman et al., Eur. Patent No.320308 (1989); and Panaccio et al., BioTechniques, 14:238-43 (1993). Thedevices use a wide variety of design principles for transfer, such aswater baths, air baths and dry blocks such as aluminum. Haff et al.,BioTechniques, 10:102-12 (1991); Findlay et al., Clin. Chem., 39:1927-33(1993); Wittwer et al., Nucl. Acids Res., 17:4353-7 (1989). PCRreactions in small reaction volumes have been described. Wittwer et al.,Anal. Biochem., 186:328-31 (1990); and Wittwer et al., Clin. Chem.,39:804-9 (1993). Polynucleotide amplification micro-devices fabricatedfrom silicon also have been described. Northrup et al., in: Digest ofTechnical Papers: Transducers 1993 (Proc. 7th International Conferenceon Solid State Sensors and Actuators) Institute of Electrical andElectronic Engineers, New York, N.Y., pp. 924-6; and Northrup et al.,PCT WO 94/05414 (1994).

Silica particles have been shown to bind to nucleic acids, and have beenused to isolate nucleic acids prior to PCR analysis. Zeillinger et. al.,BioTechniques, 14:202-3 (1993). While the art has described the use ofsilicon and other substrates fabricated with microchannels and chambersfor use in a variety of analyses, little attention has been focused onmethods for the modification of micromachined silicon or other surfaces,to diminish binding or other properties of the surfaces, which caninhibit reactions, such as polynucleotide amplification reactions,conducted in the devices. Northrup et al. describe the chemicalsilanization of a PCR reaction chamber in a silicon substrate having adepth of 0.5 mm. Northrup et al., in: Digest of Technical Papers:Transducers 1993 (Proc. 7th International Conference on Solid StateSensors and Actuators) Institute of Electrical and Electronic Engineers,New York, N.Y., pp. 924-6; and Northrup et al., PCT WO 94/05414 (1994).The reference of Northrup et al., (in: Digest of Technical Papers:Transducers 1993), however, discloses that, in the absence ofsilanization, untreated silicon surfaces of the reaction chambers had noinhibitory effect on the PCR reaction.

There is a need for convenient, rapid systems for polynucleotideamplification analyses, which could be used clinically in a wide rangeof potential applications in clinical tests such as tests for paternity,and genetic and infectious diseases and a wide variety of other tests inthe environmental and life sciences. There is a need for the developmentof micro-devices fabricated in substrates such as silicon which permitpolynucleotide amplification reactions to be conducted in high yieldswithout interfering effects on the reaction caused by the surfaces ofthe substrate.

An object of the invention is to provide microscale analytical deviceswith optimal reaction environments for conducting polynucleotideamplification reactions which can be used to detect very lowconcentrations of a polynucleotide and to produce analytical resultsrapidly. Another object is to provide easily mass produced, disposable,small (e.g., less than about 1 cc in volume) devices having functionalelements capable of rapid, automated polynucleotide amplificationanalyses of a preselected cell or cell-free sample, in a range ofapplications. It is a further object of the invention to provide agentsfor use in microscale reaction chambers fabricated in solid substratessuch as silicon, to diminish potential inhibitory effects of thesubstrate surfaces on a polynucleotide amplification reaction. It is afurther object of the invention to provide apparatus for deliveringreagents and sample fluids to and from microscale polynucleotideamplification chambers fabricated in solid substrates such as silicon,and to provide apparatus for sealing the reaction chamber during anamplification reaction. It is yet another object of the invention toprovide apparatus that can be used to implement a range of rapidclinical tests, e.g., tests for viral or bacterial infection, tests forcell culture contaminants, or tests for the presence of a recombinantDNA or a gene in a cell, and the like.

These and other objects and features of the invention will be apparentfrom the description, drawings and claims which follow.

SUMMARY OF THE INVENTION

The invention provides a family of small, mass produced, typicallyone-use devices (sometimes referred to herein as “chips”) for conductinga reaction to enable the rapid amplification of a polynucleotide in asample. In one embodiment, the device comprises a solid substrate thatis fabricated to comprise a mesoscale polynucleotide amplificationreaction chamber. The device also may include a cover, e.g., atransparent cover, disposed over the substrate, to seal at least aportion of the reaction chamber during an amplification reaction. Thedevice further includes at least one port in fluid communication withthe reaction chamber, for introducing a sample into the chamber(sometimes referred to herein as a “sample inlet port” or inlet port“).The device may include one or more flow channels extending from theports to the reaction chamber, and/or connecting two or more reactionchambers. The device also may include one or more additional ports influid communication with the reaction chamber, to serve as access ports,inlet/outlet ports and/or vents. One or more ports and/or flow channelsof the device may be fabricated in the cover or in the substrate. In thedevice, the reaction chamber may be provided with a composition whichdiminishes inhibition of a polynucleotide amplification reaction by thewall(s) defining the reaction chamber. The device may also include meansfor thermally cycling the contents of the chamber to permitamplification of a sample polynucleotide.

The term “mesoscale” is used herein with reference to reaction chambersor flow channels, at least one of which has at least one cross-sectionaldimension between about 0.1 μm and 1,000 μm. The flow channels leadingto the reaction chamber have preferred widths and depths on the order ofabout 2.0 to 500 μm. Chambers in the substrate wherein amplificationtakes place may have one or more larger dimensions, e.g., widths and/orlengths of about 1 to 20 mm. Preferred reaction chamber widths andlengths are on the order of about 0.5 to 15 mm. The reaction chambersare fabricated with depths on the order of about 0.1 to at most about1,000 μm. Typically, the reaction chambers are fabricated with depthsless than 500 μm, e.g., less than about 300 μm, and optionally less thanabout 80 μm. Fabrication of the reaction chamber, with shallow depths,e.g., less than 300 μm, advantageously facilitates heat transfer to thereaction chamber contents, e.g., through the substrate, and permitsefficient thermal cycling during an amplification reaction requiringthermal cycling. However, in some embodiments, the reaction chambers maybe fabricated with depths between about 500 μm and 1,000 μm. The overallsize of the device ranges from microns to a few millimeters inthickness, depending on the material from which it is constructed, andapproximately 0.2 to 5.0 centimeters in length or width.

The devices may be used to amplify and/or analyze microvolumes of asample, introduced into the flow system through an inlet port defined,e.g., by a hole communicating through the substrate or the cover. Thevolume of the mesoscale flow system typically will be less than 50 μl,and the volume of the reaction chambers is often less than 20 μl, e.g.,10 μl or less. The volume of the individual channels and chambers inanother embodiment may be less than 1 μl, e.g., in the nanoliter orpicoliter range. Polynucleotides present in very low concentrations,(e.g., nanogram quantities) can be rapidly amplified (e.g., in less thanten minutes) and detected. After a polynucleotide amplification assay iscomplete, the devices may be discarded or they may be cleaned andre-used.

In one embodiment, reaction chambers may be fabricated wherein the ratioof the surface area of the walls defining the reaction chamber to thevolume of the reaction chamber is greater than about 3 mm²/μl. Chambersalso may be fabricated with even higher surface area to volume ratios,such as 5 mm²/μl or, optionally, greater than 10 mm²/μl. As the ratio ofthe surface area to volume increases, heat transfer through thesubstrate to and from the reaction chamber contents is facilitated, andthus thermal cycling of the reaction becomes more efficient, and theproductivity of the reaction is increased. Additionally, however, as theratio of the surface area to volume increases, potential inhibitoryeffects of the walls of the substrate on the polynucleotideamplification reaction are increased. Depending on the material fromwhich the device is made, the wall surfaces of the mesoscale channelsand chambers could interfere with the polynucleotide amplification,e.g., via binding interactions between the material and samplepolynucleotides or amplification reagents.

The invention provides a range of compositions which may be provided inthe reaction chamber to diminish the potentially inhibitory effects ofthe reaction chamber wall surfaces, such as silicon surfaces, on thereaction. The compositions are particularly useful in reaction chambershaving a surface area to volume ratio greater than about 3 mm²/μl or 5mm²/μl, or, in another embodiment, in chambers wherein the ratio exceedsabout 10 mm²/μl. The device also may include a cover disposed over thereaction chamber to seal the reaction chamber during an amplificationreaction. The cover may comprise a material such as glass or silicon, ora plastic material. The use of a cover disposed over the reactionchamber increases the total amount of surface area in contact with fluidin the reaction chamber. The surface of the cover exposed to thereaction chamber also may be treated with compositions as disclosedherein to reduce potentially inhibitory effects of the cover surfacematerial on the amplication reaction.

A composition provided in the reaction chamber to diminish inhibition ofan amplification reaction by a wall of the reaction chamber may becovalently or non-covalently adhered to the surface of the reactionchamber wall, or may be provided in solution in the reaction chamberduring an amplification reaction. In one embodiment, the wall surfacesof one or more reaction chamber(s) and/or channel(s) in the device maybe coated with a silane, using a silanization reagent such asdimethychlorosilane, dimethydichlorosilane, hexamethyldisilazane ortrimethylchlorosilane (available, e.g., from Pierce, Rockford, Ill.).Alternatively, the surface of the walls of the reaction chamber(s)and/or the flow channel(s), e.g., fabricated within a silicon substrate,may be provided with a relatively inert coating, for example, using asiliconizing reagent, such as Aquasil™ or Surfasil™ (Pierce, Rockford,Ill.), or Sigmacote™ (Sigma Chemical Co., St. Louis, Mo.). Siliconizingreagents available from commercial manufacturers, such as Pierce(Rockford, Ill.) or Sigma Chemical Co. (St. Louis, Mo.), areorganosilanes containing a hydrolyzable group, which can hydrolyze insolution to form a silanol which can polymerize and form a film over thesurface of the chamber, and can react with hydroxyl groups on thesurface of the chamber, such that the film is tightly bonded over theentire surface. The coating may further include a macromolecule(sometimes referred to herein as a “blocking agent”) noncovalently orcovalently associated with the silicone coating, to further reduceinhibitory effects of the wall of the reaction chamber on theamplification reaction. Useful macromolecules include an amino acidpolymer, or polymers such as polyvinylpyrrolidone, polyadenylic acid andpolymaleimide.

A silicon oxide film may be provided on the surface of the reactionchamber and/or channel walls, in a silicon substrate, to reduceinhibition of the amplification reaction by the wall surfaces. Thesilicon oxide film may be formed by a thermal process wherein thesilicon substrate is heated in the presence of oxygen. Alternatively, aplasma-enhanced oxidation or plasma-enhanced chemical vapor depositionprocess may be utilized. Additionally the reaction chamber and/orchannel walls may be coated with a relatively inert polymer such as apoly (vinyl chloride).

Prior to addition of the sample polynucleotide and amplificationreagents to the reaction chamber, another polynucleotide (sometimesreferred to herein as a “blocking” polynucleotide) may be added to thechamber, such as genomic DNA or polyadenylic acid, preferably at aconcentration greater than the concentration of the samplepolynucleotide. This permits the blocking polynucleotide to occupy anysites on the wall surfaces that could potentially bind to the samplepolynucleotide and reduce the yield of the reaction or the precision ofthe assay. Thus, in one-embodiment, a blocking polynucleotide may beprovided in a reaction chamber fabricated within a silicon substrate,such that the blocking polynucleotide may occupy any polynucleotidebinding sites, such as free hydroxyl groups, on the wall surfaces of thereaction chamber. To avoid interfering with the amplification reaction,the blocking polynucleotide should comprise sequences unrelated to thatof the sample polynucleotide. Other compositions which bind to thechamber wall surfaces, such as polyguanylic acid or various polypeptidessuch as casein or serum albumin, could also be utilized as a blockingagent.

The devices may be utilized to implement a polynucleotide amplificationreaction, such as a polymerase chain reaction (PCR), in the reactionchamber. The reaction chamber may be provided with reagents for PCRincluding a sample polynucleotide, polymerase, nucleoside triphosphates,a first primer hybridizable with the sample polynucleotide, and a secondprimer hybridizable with a sequence that is complementary to the samplepolynucleotide, wherein the first and second primers define the terminiof the amplified polynucleotide product. The device also may includemeans for thermally cycling the contents of the amplification reactionchamber, such that, in each cycle, e.g., the temperature is controlledto 1) dehybridize (“melt”) double stranded polynucleotide, 2) anneal theprimers to single stranded polynucleotide, and 3) synthesize amplifiedpolynucleotide between the primers. Other amplification methodsavailable in the art also may be utilized, including, but not limitedto: (1) target polynucleotide amplification methods such asself-sustained sequence replication (3SR) and strand-displacementamplification (SDA); (2) methods based on amplification of a signalattached to the target DNA, such as “branched chain” DNA amplification(Chiron Corp.); (3) methods based on amplification of probe DNA, such asligase chain reaction (LCR) and QB replicase amplification (QBR); and(4) various other methods such as ligation activated transcription(LAT), nucleic acid sequence-based amplification (NASBA), repair chainreaction (RCR) and cycling probe reaction (CPR) (for a review of thesemethods, see pp. 2-7 of The Genesis Report, DX, Vol. 3, No. 4, February1994; Genesis Group, Montclair, N.J.).

The reaction chamber may be fabricated with one section which isthermally cycled sequentially between the required temperatures forpolynucleotide amplification reactions requiring thermal cycling, suchas conventional PCR. Alternatively, the reaction chamber may comprisetwo or more sections, set at the different temperatures required fordehybridization, annealing and polymerization, in which case the devicefurther comprises means for transferring the contents of the chamberbetween the sections to implement the reaction, e.g., a pump controlledby a computer. The reaction chamber may be bound in at least a portionof the chamber by a cover disposed over the substrate. The device mayfurther include means for detecting the amplified polynucleotide, asdisclosed herein. The devices may be used to implement a variety ofautomated, sensitive and rapid polynucleotide analyses, includinganalyses for the presence of polynucleotides in cells or in solution, orfor analyses for a virus or cell types using the presence of aparticular polynucleotide as a marker.

The mesoscale flow channel(s) and reaction chamber(s) may be designedand fabricated from solid substrates using established micromachiningmethods such as photolithography, etching and disposition techniques,laser machining, LIGA processing ('Becker et al., Microelec. Eng. 4:35-56, 1986) and plastic molding. The mesoscale flow systems in thedevices may be constructed by fabricating flow channels and one or morereaction chambers into the surface of the substrate, and then adheringor clamping a cover over the surface. The solid substrate and/or covermay comprise a material such as silicon, polysilicon, silica, glass,gallium arsenide, polyimide, silicon nitride and silicon dioxide. Thecover and/or the substrate alternatively may comprise a plastic materialsuch as an acrylic, polycarbonate polystyrene or polyethylene.Optionally the cover and/or substrate may comprise a transparentmaterial.

An appliance also may be provided, for use with the device, whichcontains a nesting site for holding the substrate of the device andwhich optionally mates one or more input ports on the substrate with oneor more flow lines in the appliance. After a biological fluid samplesuspected to contain a particular polynucleotide is applied to the inletport, the substrate is placed in the appliance and pumps, e.g., disposedin the appliance, are actuated to force the sample through the flowsystem. Alternatively, a sample may be injected into the substrate bythe appliance (e.g. by a syringe fitted to the appliance). Reagentsrequired for the assay, such as a polymerase enzyme, may be added (inliquid or in dry form) to the polynucleotide sample prior to injectioninto the substrate. Alternatively, reagents necessary to complete theassay can be injected into the reaction chamber from a separate inletport, e.g., by the appliance. Fluid samples and reagents may also enterthe mesoscale flow system by capillary action or by gravity.

The invention also provides means for sealing one or more of the fluidinlet/outlet ports in the device during an amplification reaction. Thisadvantageously prevents evaporation of liquids during thermal cyclingand thus maintains the preferred reaction concentrations during theamplification reaction. In one embodiment, an apparatus including meansfor delivering fluid to and from the reaction chamber through a port inthe device, and adapted to interfit and/or interlock with the port isprovided, which'can reversibly seal the port after delivery of fluid tothe reaction chamber. For example, the fluid delivery apparatus maycomprise a syringe or pipette. In one embodiment, the fluid deliveryapparatus may comprise a pipette including a pipette tip provided withan aperture for transferring fluid between the pipette and the port. Thepipette tip optionally may be releasable from the pipette, and may bedisposable to prevent contamination between samples.

The device may include a substrate comprising a heat conducting materialsuch as silicon, as well as a cover disposed over the substrate, whichmay comprise a transparent material such as glass or a plastic. Thedevice also includes the mesoscale polynucleotide amplification chamber,fabricated within the substrate or the cover. The cover may include acavity for receiving and interfitting with the pipette used to deliversample and reagent solutions to and from the reaction chamber. Thedevice may further include a flow channel that communicates through thesubstrate and/or the cover between the aperture of the pipette tip andthe reaction chamber, when the pipette is fitted within the cavity. Theaperture may be positioned on a wall of the pipette tip to permit thepipette tip to move between a first position which permits transfer offluid from the tip through the aperture and the channel to the reactionchamber, and a second position to permit the aperture to face a wall ofthe cavity, thereby to seal the flow channel and the reaction chamberduring a reaction. Additionally, a depressible member may be providedwhich extends from the substrate and can seal the port upon depressionof the member against the port.

The temperature of one or more section(s) in the reaction chamber can beregulated by, e.g., providing one or more electrical resistance heatersin the substrate near the reaction chamber, or by using a pulsed laseror other source of electromagnetic energy directed to the reactionchamber. The appliance may include electrical contacts in the nestingregion which mate with contacts integrated into the structure of thesubstrate, e.g., to power electrical resistance heating of the reactionchamber. A cooling element may also be provided in the appliance, toassist in the thermal regulation of the reaction chamber. The appliancemay be provided with conventional circuitry in communication withsensors in the device for thermally regulating the temperature cyclesrequired for the dehybridization and polymerization reactions.

The amplified polynucleotide produced by the polynucleotideamplification reaction in the mesoscale reaction chamber can becollected through a port in the substrate and detected. Alternatively,specific reagents and methods known in the art may be employed todirectly detect amplification products in the reaction chamber (“TaqMan”™ PCR reagents and kit, available from Perkin Elmer Corp., forexample). As another alternative, a mesoscale detection region may bemicrofabricated in the substrate, in fluid communication with thereaction chamber in the device, as a part of the mesoscale flow system.The detection region may include a labeled binding moiety, such as alabeled polynucleotide or antibody probe, capable of detectably bindingwith the amplified polynucleotide. The presence of polymerizedpolynucleotide product in the detection region can be detected, e.g., byoptical detection of agglutination of the polymerized polynucleotide andthe binding moiety through a glass cover over the detection region orthrough a translucent or transparent section of the substrate itself.Alternatively, the detection region may comprise a series of channels ormicrolithographic arrays for electrophoretically separating anddetecting an amplified polynucleotide.

A positive assay may also be indicated by detectable changes in samplefluid flow properties, such as changes in pressure or electricalconductivity at different points in the flow system upon production ofamplified polynucleotide in the reaction chamber. In one embodiment, thedevice comprises a mesoscale flow system which includes a polynucleotideamplification reaction chamber, and a detection region (e.g., a chamberor a portion of a flow channel), used in combination with an appliancewhich includes sensing equipment such as a spectrophotometer capable ofreading a positive result through an optical window, e.g., disposed overthe detection region. The appliance may also be designed to receiveelectrical signals indicative of a pressure reading, conductivity, orthe like, sensed in the reaction chamber, the detection region, or someother region of the flow system.

The substrate may comprise a plurality of reaction and/or detectionchambers to enable the rapid parallel amplification and/or detection ofseveral polynucleotides in a mixture. The mesoscale flow system mayinclude protrusions, or a section of reduced cross-sectional area, tocause lysis of cells in the microsample prior to delivery to thereaction chamber. Sharp edged pieces of silicon, trapped in the flowpath, can be used as a lysis means. The mesoscale flow system also mayinclude a cell capture region comprising a binding moiety, e.g.,immobilized on a wall of a flow channel, which binds a particular typeof cell in a heterogeneous cell population at a relatively low fluidflow rate, and at a greater flow rate or by changing the nature of thesolvent, for example, releases the cell type prior to delivery of thecells to a cell lysis region, then to a reaction chamber. In thisembodiment, intracellular DNA or RNA is isolated from a selected cellsubpopulation and delivered to the mesoscale reaction chamber forpolynucleotide analysis in one device. In an alternative embodiment, thebinding reagent may by immobilized on a solid particle, such as a latexor magnetic bead, as described below.

Complex-forming agents, such as magnetic beads coated with apolynucleotide probe, may be provided within the mesoscale flow system,which can be moved along the flow system by an external magnetic field,e.g., in the appliance. The polynucleotide probe immobilized on themagnetic beads enables the beads to bind to amplified polynucleotide inthe reaction chamber or in a separate detection chamber. Magnetic beadscontaining an immobilized polynucleotide probe may be, e.g., carriedthrough the flow system or otherwise introduced to the reaction chamberat the end of an assay to bind to the amplified polynucleotide product.The bound polynucleotide may then be transported on the magnetic beadsto a detection or purification chamber in the flow system, or to acollection port. Alternatively, the magnetic beads may be held in placeat a predetermined location in the device, then transported to adetection or purification chamber after binding the polynucleotideproduct.

Some of the features and benefits of the devices are illustrated inTable 1. The devices can provide a rapid test for the detection ofpathogenic bacteria or viruses, or for the presence of certain celltypes, or the presence of a gene or a recombinant DNA sequence in acell. The devices as disclosed herein are all characterized by amesoscale flow system including a polynucleotide amplification reactionchamber, preferably having at least one mesocale dimension, which isused to amplify a polynucleotide in a sample, and which may be providedwith the required amplification reagents. The device may be used toamplify a polynucleotide in a wide range of applications. At theconclusion of the assay the device may be discarded, or it may becleaned and re-used.

TABLE 1 Feature Benefit Flexibility No limits to the number of devicedesigns or applications available. Reproducible Allows reliable,standardized, mass production of devices. Low Cost Allows competitivepricing with Production existing systems. Disposable nature forsingle-use processes. Small Size No bulky instrumentation required.Lends itself to portable units and systems designed for use in non-conventional lab environments. Minimal storage and shipping costs.Microscale Minimal sample and reagent volumes required. Reduces reagentcosts, especially for more expensive, specialized test procedures.Allows simplified instrumentation schemes. Sterility Devices can besterilized for use in microbiological assays and other proceduresrequiring clean environments. Sealed System Minimizes biohazards.Ensures process integrity. Multiple Circuit Can perform multipleprocesses or Capabilities analyses on a single device. Allows panelassays. Multiple Expands capabilities for assay and Detector processmonitoring to virtually Capabilities any system. Allows broad range ofapplications. Reusable Devices Reduces per process cost to the user forcertain applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic longitudinal cross-sectional view of a device 10according to the invention that includes a solid substrate 14, on whichis machined mesoscale flow channel 20 connected to inlet ports 16 andpolynucleotide amplification reaction chamber 22, with a cover 12adhered to the surface of the substrate.

FIG. 1B is a schematic longitudinal cross-sectional view of analternative embodiment device 10 according to the invention thatincludes a solid substrate 14, on which is machined the mesoscalepolynucleotide amplification reaction chamber 22 and inlet ports 16,with cover 12 adhered to the surface of the substrate.

FIG. 1C is a schematic longitudinal cross-sectional view of anotherembodiment of device 10 which includes a solid substrate 14 fabricatedwith mesoscale polynucleotide amplification reaction chamber 22, andcover 12 fabricated with ports 16 and channels 20 in fluid communicationwith chamber 22.

FIG. 2A is a perspective view of the device of FIG. 1A.

FIG. 2B is a perspective view of the device of FIG. 1B.

FIG. 3A is a schematic illustration of an analytical device 10 nestedwithin a schematically illustrated appliance 50, which may be used tosupport the device 10 and which includes heating element 57 forregulating the temperature of the reaction chamber 22 in device 10.

FIG. 3B is a schematic illustration of an analytical device 10 nestedwithin appliance 50, which may be used to support the device 10 andwhich includes the heating element 53 for regulating the temperature ofthe reaction chamber 22 in device 10.

FIG. 4 is a schematic longitudinal cross-sectional view of a deviceaccording to the invention that includes a solid substrate 14, on whichis machined mesoscale flow channel 20 connected to inlet ports 16 andreaction chamber sections 22, with a cover 12 adhered to the surface ofthe substrate.

FIG. 5 is a perspective view of the device of FIG. 4.

FIG. 6A is a schematic illustration of analytical device 10 nestedwithin appliance 50, which may be used to support the device 10, andwhich includes heating elements 57 for regulating the temperature of thereaction chamber sections 22 in device 10.

FIG. 6B is a schematic illustration of analytical device 10 nestedwithin appliance 50, which may be used to support the device 10 andwhich includes heating element 57 for regulating the temperature of thereaction chamber section 22A in device 10.

FIG. 7 is a schematic plan view of a substrate 14 microfabricated withmesoscale reaction chamber sections 22A and 22B, in fluid communicationwith a detection chamber comprised of a diverging system of flowchannels 40 of progressively decreasing cross-sectional dimensiondisposed on the substrate.

FIG. 8 is a cross sectional perspective view of a flow channel 20 insubstrate 14 with cell or debris filtering protrusions 80 extending froma wall of the channel.

FIG. 9 is a cross sectional perspective view of a flow channel 20 insubstrate 14 with cell piercing protrusions 90 extending from a wall ofthe channel.

FIG. 10A is a schematic plan view of a mesoscale analytical deviceincluding reaction chamber sections 22A and 22B, and detection chamber22C, microfabricated in the substrate 14.

FIG. 10B is a schematic plan view of another mesoscale analytical deviceincluding reaction chamber sections 22A and 22B, and detection region26, microfabricated in the substrate 14.

FIG. 11 is a schematic plan view of another mesoscale analytical deviceincluding a reaction chamber 22A microfabricated in the substrate 14.

FIG. 12 is a schematic plan view of an analytical device fabricated witha series of mesoscale chambers suitable for implementing a variety offunctions including cell sorting, cell lysing and polynucleotideanalysis.

FIG. 13 is a schematic plan view of an analytical device fabricated withtwo systems of split flow channels 40.

FIGS. 14, 15 and 16 illustrate top plan views of different embodimentsof a mesoscale filter 24 microfabricated in flow channel 20 in ananalytical device 10.

FIG. 17 is a schematic perspective view of an apparatus 60 used incombination with device 10 (not shown) for viewing the contents ofdevice 10.

FIG. 18 is a schematic cross-sectional view of the apparatus 60 of FIG.17.

FIG. 19 is a schematic cross-sectional view of a device includingsubstrate 14 and transparent cover 12 which includes cavity 87 receivingpipette 86.

FIG. 20 is a schematic cross-sectional view of a pipette tip 84including aperture 88.

FIG. 21 is a schematic cross-sectional view of a substrate 14 providedwith, member 85 which may be compressed to seal port 16 and channel 20.

FIG. 22 is a schematic perspective view of an apparatus includingtransparent cover 12 provided with cavity 87 and flow passage 20Aleading to flow channel 20B and reaction chamber 22 in substrate 14.

FIG. 23 is a drawing illustrating an agarose gel electrophoresis patternof polynucleotide samples amplified in a mesoscale amplificationchamber.

Like reference characters in the respective drawn figures indicatecorresponding parts. The drawings are not necessarily to scale.

DETAILED DESCRIPTION A. General

The invention provides a family of small, mass produced, typicallyone-use devices for performing polynucleotide amplification reactions toenable the rapid amplification of polynucleotides in fluid samples. Thedevice comprises a solid substrate, fabricated to include at least onepolynucleotide amplification reaction chamber, and typically is of alength and/or width ranging from approximately 0.1 to 5.0 centimeters:The channels and chambers in cross-section through the thickness of thedevice may be triangular, truncated conical, square, rectangular,circular, or any other shape. The device also includes a sample inletport in fluid communication with the reaction chamber. The device alsomay include additional ports (which may function as access orinlet/outlet ports, or as vents) disposed at any location over the flowsystem, and one or more sample flow channels, in fluid communicationwith the reaction chamber. One or more of the port(s) may be open to theatmosphere or attached to appropriate pressure or suction devices, e.g.for filling or evacuating the device, or they may be sealed, e.g. duringan amplification reaction. The port(s) and channel(s) may be fabricatedin the substrate or, alternatively, in a cover disposed over thesubstrate, or both. The device may further include a system forthermally cycling the contents of the reaction chamber to permitamplification of a sample polynucleotide.

At least one of the reaction chambers and the flow channels of thedevice, and preferably both, have a mesoscale dimension, i.e., at leastone cross-sectional dimension on the order of 0.1 to 1,000 μm. Thepreferred depth of the reaction chamber is less than about 500 μm, morepreferably less than 300 μm and most preferably less than 80 μm. Thereaction chambers may have larger widths and lengths, e.g., on the orderof about 1-20 mm, preferably about 5-15 mm.

The shallow depth of the reaction chamber advantageously facilitatesheat transfer to the reaction chamber contents, e.g., from a heaterpositioned near the substrate, and permits efficient thermal cyclingduring an amplification reaction. In one embodiment, the reactionchamber may be fabricated such that the ratio of the surface area of thewalls of the reaction chamber to the volume of the reaction chamberrange from about 3 mm²/μl to greater than about 10 mm²/μl. As the ratioof the surface area to volume increases, heat transfer through thesubstrate and the effectiveness of the thermal cycling of the reactionis improved. However, potential inhibitory effects of the walls of thesubstrate on the amplification reaction also maybe increased, dependingon the material from which the substrate is constructed. Accordingly,compositions are provided which are useful in diminishing inhibitoryeffects of wall surfaces, such as silicon surfaces, in reaction chambersin which such treatment is warranted.

Compositions provided to diminish inhibition of an amplificationreaction by a wall of the reaction chamber may be covalently ornon-covalently adhered on the chamber surface. Alternatively, acomposition may be provided in solution in the reaction chamber duringan amplification reaction. In one embodiment, the mesoscale flowchannel(s) and reaction chambers may be fabricated in a siliconsubstrate. The walls of the reaction chamber(s) and/or channel(s) thenmay be coated with a composition which reduces inhibition of thereaction by the silicon surfaces in the device. For example, the siliconsurfaces of the device may be coated with any of a range of silanizingreagents available in the art as disclosed herein.

In one embodiment, the devices of the invention may be utilized toconduct a polymerase chain reaction (PCR) in the reaction chamber. Thechamber is provided with reagents required for a polymerase chainreaction including the sample polynucleotide, a polymerase such as Taqpolymerase, nucleoside triphosphates, a first primer hybridizable withthe sample polynucleotide, and a second primer hybridizable with asequence complementary to the polynucleotide, wherein the first andsecond primers define the termini of the polymerized productpolynucleotide. Reagents may be added to a sample and then deliveredthrough an inlet port to the mesoscale reaction chamber, or the reagentsmay be delivered to the reaction chamber independently of the samplethrough a separate inlet port.

The polymerase chain reaction may be performed, according to methodsestablished in the art (Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, 1989). Any thermostablepolynucleotide polymerase available in the art may be utilized. Thedevice may include means for thermally cycling the contents of thechamber such that, in each cycle, temperature is controlled todehybridize double stranded polynucleotide to produce single strandedpolynucleotide, and then to anneal the primers and enable polynucleotidepolymerization.

Although polynucleotide amplification by the polymerase chain reactionhas been described and exemplified herein, it will be appreciated bypersons skilled in the art that the devices and methods of the presentinvention may be utilized equally effectively for a variety of otherpolynucleotide amplification reactions. Such additional reactions may bethermally dependent, such as the polymerase chain reaction, or they maybe carried out at a single temperature (e.g., nucleic acidsequenced-based amplification (NASBA)). Moreover, such reactions mayemploy a wide variety of amplification reagents and enzymes, includingDNA ligase, T7 RNA polymerase and/or reverse transcriptase, amongothers. Additionally, denaturation of polynucleotides can beaccomplished by known chemical or physical methods, alone or combinedwith temperature change. Polynucleotide amplification reactions that maybe practiced in the device of the invention include, but are not limitedto: (1) target polynucleotide amplification methods such asself-sustained sequence replication (3SR) and strand-displacementamplification (SDA); (2) methods based on amplification of a signalattached to the target polynucleotide, such as “branched chain” DNAamplification (Chiron Corp. Emeryville, Calif.); (3) methods based onamplification of probe DNA, such as ligase chain reaction (LCR) and QBreplicase amplification (QBR); (4) transcription-based methods, such asligation activated transcription (LAT) and nucleic acid sequence-basedamplification (NASBA); and (5) various other amplification methods, suchas repair chain reaction (RCR) and cycling probe reaction (CPR) (for asummary of these methods and their commercial sources, see pp. 2-7 ofThe Genesis Report, DX, Vol. 3, No. 4, February 1994; Genesis Group,Montclair, N.J.).

The capacity of the devices of the invention is small, enabling assaysto be performed on very small amounts of a liquid sample (e.g., lessthan 50 μl and preferably less that 10 μl). The mesoscale flow systemsof the devices may be microfabricated with microliter volumes, oralternatively nanoliter volumes or less, which advantageously limits theamount of sample and/or reagent fluids required for an assay. Thedevices may be used to implement a variety of automated, sensitive andrapid polynucleotide analyses including the analysis of polynucleotidesin cells or in solution. At the conclusion of the assay the devices maybe cleaned and re-used, or discarded. The use of disposable deviceseliminates contamination and reduces biohazards. The sample and reactionmixture at all times can remain entombed, and the low volume simplifieswaste disposal.

B. Substrate Fabrication

Analytical devices comprising a solid substrate and optionally, a coverdisposed over the substrate, can be designed and fabricated withmesoscale flow channels and reaction chambers from a wide range ofmaterials. The devices optionally may be fabricated from a materialwhich can be sterilized easily. Silicon provides a useful materialbecause of the well-developed technology permitting its precise andefficient fabrication, but a wide range of other materials may be usedwithin the scope of the invention. Other materials which may be utilizedinclude, e.g., gallium arsenide, indium phosphide, aluminum,polysilicon, silicon nitride, silicon dioxide, polyimide andthermocouple materials such as chrome/aluminum, as well as quartz,glass, diamond, polycarbonate, polystyrene and other polymers such aspolytetrafluoroethylenes. Other possible materials include superalloys,zircaloy, steel, gold, silver, copper, tungsten, molybdenum, tantalum,KOVAR, ceramics, KEVLAR, KAPTON, MYLAR, brass, sapphire, or any of arange of plastics and organic polymeric materials available in the art.

The port(s), mesoscale flow system, including sample flow channel(s) andreaction chamber(s), and other functional elements may be fabricatedinexpensively in large quantities from, e.g., a silicon substrate by anyof a variety of micromachining methods known to those skilled in theart, Micromachining methods available include film deposition processessuch as chemical vapor deposition, laser-based fabrication orphotolithographic techniques such as UV, X-ray, LIGA processes andplastic molding, or etching methods which may be performed by either wetchemical processes or plasma processes. (See, e.g., Manz et al., Trendsin Analytical Chemistry, 10:144-149 (1991)). The arrangement ofchannels, chambers, and multiple ports facilitates the sequential,properly timed, and volumetrically correct addition of sample andreagents within the device.

In one embodiment, flow channels or chambers of varying widths anddepths can be fabricated, with at least one having a mesoscaledimension, in a substrate such as silicon. The substrate containing afabricated mesoscale flow channel and reaction chamber may be coveredand sealed with a glass cover clamped, anodically bonded or otherwiseadhered to the substrate. Other clear or opaque cover materials may beused. Alternatively, two substrates can be sandwiched, or a substratecan be sandwiched between two glass covers. The use of a transparentcover results in a window which facilitates dynamic viewing of contentsin the mesoscale flow system. Other fabrication approaches may be used.

C. Passivation Methods

A composition may be provided in the mesoscale amplification reactionchamber or flow channel to passivate the wall surfaces, i.e., todiminish inhibition of the amplification reaction by the wall surfacesif the nature of the wall material necessitates such treatment. Thecomposition may be adhered to the surface of the reaction chamber orchannel walls, either covalently or non-covalently. For example, thewall surfaces may be coated with any of a range of silanization agentsknown in the art. Alternatively, the composition may be provided in thechamber in solution, together with the sample polynucleotide and theamplification reagent during an analysis. Mesoscale reaction chambersmay be fabricated wherein the ratio of the surface area of the walldefining the reaction chamber to the volume of the chamber ranges fromabout 3 mm²/μl to greater than 10 mm²/μl, or, optionally, greater than20 mm²/μl. As the surface area to volume ratio increases, heat transferto the reaction chamber through the substrate is improved, and athermally dependent amplification reaction may be cycled more rapidly.Concurrently, however, inhibitory effect of the wall surfaces may alsobe enhanced as the ratio of surface area to volume increases. Thecompositions for reducing inhibition of the amplication reaction by awall of the reaction chamber are particularly useful in chambers with ahigh surface area to volume ratio, e.g., greater than about 3 mm²/μl.

It will be appreciated by those skilled in the art that the passivationcompositions and methods described herein are applicable to only certainmaterials wherein it has been observed that amplification reactions maybe improved by passivating reaction chamber surfaces. Some materialscontemplated for use in devices of the invention are naturally inert,and so would not benefit from the passivation treatments describedherein.

The substrate may comprise a heat conductive material such as silicon orglass. The reaction chamber and/or channel walls may be passivated bycoating the surface with a silane using a silanization agent availablein the art. Useful silanization agents include dimethylchlorosilane(DMCS), dimethyldichlorosilane (DMDCS), hexamethyldisilazane (HMDS), andtrimethylchlorosilane (TMCS). These chlorosilanes can react covalentlywith surface hydroxyl groups on the walls comprising silicon or anothermaterial that potentially could interfere with the reaction by, e.g,binding to the sample polynucleotide or the amplification reagents.

Additionally, the walls of the reaction chambers and/or channels may beprovided with a silicone coating using a commercially availablesiliconizing reagent, such as Aquasil™ or Surfasil™ (Pierce, Rockford,Ill.), or Sigmacote™ (Sigma Chemical Co., St. Louis, Mo.). Siliconizingreagents available from commercial manufacturers, such as Pierce(Rockford, Ill.) or Sigma Chemical Co. (St. Louis, Mo.), areorganosilanes containing a hydrolyzable group, which can hydrolyze insolution to from a silanol which can polymerize and form a film over thesurface of the chamber, and can react with hydroxyl groups on thesurface of the chamber, such that the film is tightly bonded over theentire surface of the chamber.

The coating may further include a macromolecule noncovalently orcovalently associated with the coating, to further reduce inhibitoryeffects of the wall of the reaction chamber on the amplificationreaction. Useful macromolecules include an amino acid polymer, orpolymers such as polyvinylpyrrolidone, polyadenylic acid, orpolymaleimide or compositions such as maleimide. Other usefulmacromolecules include, e.g., poly-L-alanine, poly-L-aspartic acid,polyglycine, poly-L-phenylalanine, or poly-L-tryptophan. A silicon oxidefilm may be provided on the reaction chamber and/or channel walls toreduce inhibition of the amplification reaction by silicon wallsurfaces. The silicon oxide film may be formed by a thermal processwherein the substrate is heated in the presence of oxygen.Alternatively, a plasma-enhanced oxidation or chemical vapor depositionprocess may be utilized. Additionally, the reaction chamber and/orchannel walls may be coated with polymer, such as poly(vinyl chloride).For example, a solution of poly(vinyl chloride) in chloroform may beadded to the mesoscale flow system, and then the coating may be formedupon evaporation of the solvent.

In another embodiment, a blocking agent, such as a polynucleotide orpolypeptide, may be added to the chamber. For example, genomic DNA orpolyadenylic acid may be added to the solution in the reaction chamber,at a concentration preferably greater than the concentration of thesample polynucleotide. This permits the polynucleotide to occupy anysites on the wall surfaces that potentially could bind to the samplepolynucleotide or assay reagents andreduce the yield of the reaction. IfDNA or RNA is used as the blocking polynucleotide, it should beeffectively devoid of sequences that could interfere with theamplification reaction (i.e., it should contain substantially onlysequences unrelated to those of the sample polynucleotide). Othercompositions which could be utilized as blocking agents include bovineserum albumin or an amino acid polymer, or polymers such aspolyvinylpyrrolidone, or polymaleimide or compositions such asmaleimide.

D. Thermal Cycling

A polynucleotide amplification reaction, such as a PCR reaction, may beconducted in the reaction chamber of the device 10 shown in FIGS. 1A and2A. An alternative embodiment of device 10 is illustrated in FIGS. 1Band 2B. As illustrated schematically in FIGS. 1A, 1B, 2A and 2B, thedevice 10 may include a silicon substrate 14 microfabricated with inletports 16, a mesoscale flow channel 20, and reaction chamber 22. Thepolynucleotide sample and the reagents required for the polymerizationreaction are added, and the products withdrawn (if necessary) throughflow channel 20 from reaction chamber 22 through inlet ports 16 whichare fabricated on one end of the flow channel 20. The substrate 14 iscovered, e.g., with a glass or plastic cover 12. The device 10 may beused in combination with an appliance, such as appliance 50 shownschematically in FIG. 3A. Appliance 50 includes a nesting site 58 forholding the device 10, and for registering ports, e.g., ports 16 ondevice 10, with a flow line 56 in the appliance. A pump 52 in appliance50 is used to deliver a sample and/or reagents from flow line 56 in theappliance to the reaction chamber 22 via the inlet ports 16.

The appliance 50 may include a heating/cooling element 57 forcontrolling the temperature within the PCR chamber, e.g., an electricalheating element and/or a refrigeration coil. The electrical heatingelement may alternatively be integrated into the substrate 10, withcontacts for power mated to matching electrical contacts in theappliance below the reaction chamber 22. Alternatively, as shown in FIG.3B, the appliance may include a heating means 53, such as a laser, aPeltier heater, or a source of electromagnetic energy, disposed over oradjacent to the reaction chamber in device 10. The heater also may bedisposed in the appliance below the reaction chamber. A microprocessorin the appliance may be used to regulate the heating element in order toprovide a temperature cycle in the amplification chamber between atemperature suitable for dehybridization, e.g., 94° C., and atemperature suitable for annealing and polymerization, e.g., 40-60° C.for annealing and 70-75° C. for polymerization. A thermocouple,thermistor or resistance thermometer may also be provided in thesubstrate in electrical contact with the appliance, to allow themicroprocessor to detect and maintain the temperature cycles in thereaction chamber. Heating and sensing can advantageously be combined byusing a single element, e.g. resistance thermometer, for both purposes,combining heating and sensing either simultaneously or on a multiplexedbasis.

A cooling element, such as a miniature thermoelectric heat pump(Materials Electronic Products Corporation, Trenton, N.J.), Peltierthermocouple or Joule Thompson cooling device, may also be included inthe appliance for adjusting the temperature of the reaction chamber. Inanother embodiment, in the appliance shown in FIG. 3B, the temperatureof the reaction chamber can be regulated by a timed laser pulse directedat the reaction chamber through glass cover 12, so as to allowsequential heating and cooling of the sample to the requiredtemperatures for the polynucleotide amplification cycle. Additionally,heating and cooling can be advantageously combined by the use of Peltierthermocouples to provide both these functions. The thermal properties ofsilicon enable a rapid heating and cooling cycle. The use of reactionchambers fabricated with a high surface area to volume ratio, e.g.,greater than 3 mm²/μl, is advantageous, since heat transfer to and fromthe reaction chamber contents is facilitated. This enhances theefficiency of thermal cycling and the productivity of the amplificationreaction within the chamber.

As illustrated schematically in FIGS. 4, 5 and 6A, a mesoscalepolynucleotide amplification reaction chamber may be microfabricatedwith multiple sections, e.g., two sections 22A and 22B, connected byflow channel 20B. In this embodiment, section 22A is heated to ormaintained at a temperature suitable for dehybridization and section 22Bis heated to or maintained at a temperature suitable for annealing andpolymerization. During an analysis, the device 10 may be placed inappliance 50 (FIG. 6A). The appliance 50 is provided with means 57 forcontrolling the temperature of the reaction chamber sections.Alternatively, a laser may be used to heat the sections. A thermocoupleor other temperature sensing device can be included in the substrate tomonitor the temperatures of the sections of the reaction chamber, andits output may be used to control thermal input, e.g., with the aid of amicroprocessor.

In operation, a pump 52 in the appliance is used to deliver thepolynucleotide sample and the required reagents from flow line 56through inlet port 16A to section 22A. The pump 52, which also may becontrolled by a microprocessor in the appliance, is then used totransfer the sample periodically, between sections 22A and 22B, throughchannel 20B to implement a repetitive polynucleotide amplificationreaction cycle, while port 16B serves as a vent. When the reaction iscomplete, the pump 52 in appliance 50 may be used to deliver the samplethrough port 16B and line 56 in the appliance to port 59 to recover theproduct. Of course, three or more chambers may be used, each of which ismaintained at a temperature suitable for conducting a particularreaction.

In the device 10 shown in FIGS. 4, 5 and 6B, a heating element may beused to heat section 22A to a temperature suitable for dehybridizationof double stranded DNA, e.g., 94° C., while section 22B and channel 20B,which connects sections 22A and 22B, are spaced apart from section 22Asuch that upon transport of a heated sample from section 22A to section22B, heat is dissipated sufficiently to permit the temperature of thesample to fall to the temperature required for annealing andpolymerization before the sample is returned to section 22A for furthercycling. This may be achieved readily as silicon has a relatively highthermal conductivity and the area of interface between the liquid sampleand the substrate is quite high. In this embodiment, microprocessors inthe appliance 50 are used to control pump 52, which regulates the flowcycle of the sample between sections 22A and 22B. Thus, a dynamicthermal equilibrium creates a temperature gradient along the flow pathbetween the chambers, and appropriate temperatures are achieved in bothusing a single heating source. Other designs are possible. For example,the annealing and polymerization reactions could be implemented indifferent sections of a single chamber, set at different optimizedtemperatures.

E. Sealing Fluid Transfer Ports

The devices include a solid substrate fabricated with a mesoscalepolynucleotide amplification chamber. The devices further include atleast one sample inlet port, and a sample flow channel connecting theinlet port to the reaction chamber. One or more ports and flow channelsin the device may be fabricated within the substrate (FIG. 1A) or in acover disposed over the substrate (FIG. 1C). The cover may comprise,e.g., a transparent material, such as glass or any of a range of plasticmaterials available in the art.

The invention provides means for sealing one or more of the ports duringan amplification reaction, to prevent evaporation of liquids duringthermal cycling. In one embodiment, a fluid delivery apparatus isprovided for delivering fluid to and from the reaction chamber throughthe port, which is adapted to interfit with and/or interlock with theport, and which can reversibly seal the port after delivery of fluid tothe reaction chamber. A syringe or pipette capable of interfitting withand sealing a fluid entry/exit port in the substrate may be utilized.

As illustrated in FIGS. 19 and 22, in one embodiment, cover 12 may befabricated with cavity 87 for interfitting with and receiving a pipette86. Pipette 86 may be provided with a pipette tip 84 which includes anaperture 88 for transferring fluid from the pipette tip 84 through flowchannel 20A in the cover to flow channel 20B and amplification reactionchamber 22 in substrate 14, when the pipette is interfitted in thecavity 87. The pipette tip optionally may be releasable from thepipette, and may be disposable to prevent contamination between samples.

As illustrated in FIG. 20, the aperture 88 may be positioned on a sidewall of pipette tip 84, to permit the pipette tip, on a pipetteinterfitted in cavity 87 in device 10 shown in FIG. 22, to move betweena first position which permits transfer of fluid from the tip throughthe aperture 88 to the flow channel 20A and to the reaction chamber 22,and a second position to permit the aperture to face a wall of thecavity 87, thereby to seal the channel 20A and the chamber 22 during areaction. Additionally, a depressible member 85 may be provided, whichextends from the substrate, and which is capable of sealing the portupon depression of the member 85, as illustrated in FIG. 21.

Devices comprising sealed fluid transfer ports as described above may beutilized for a variety of purposes other than polynucleotideamplification. For example, such ports may be employed in a separatedevice for sample preparation, immunoassay, or both, as described incommonly owned co-pending Application Serial No. [not yet assigned] thedisclosure of which has been incorporated herein by reference.

F. Detection of Amplified Polynucleotide

Amplified polynucleotide present in the reaction chamber may be detectedby a range of methods known in the art for detecting polynucleotides,such as electrophoresis in an agarose gel in the presence of ethidiumbromide. In one embodiment, the amplified polynucleotide product may bedetected directly in the reaction chamber, using commercially availablereagents developed for that purpose (e.g., “Taq Man”™ reagents, PerkinElmer Corporation). The devices also may be provided with a means fordetecting amplified polynucleotide disposed either in the substrate orin an appliance used in combination with the substrate. The presence ofamplified polynucleotide product in the device can be detected by any ofa number of methods including, but not limited to: (1) monitoring thepressure or electrical conductivity of sample fluids entering and/orexiting the reaction chamber in the mesoscale flow system; (2) forming adetectable complex by, e.g., binding the polynucleotide product with alabeled probe, such as a labeled oligonucleotide or antibody probe; and(3) electrophoretically separating the polynucleotide product fromreactants and other components of the sample.

The analytical devices also may be utilized in combination with anappliance for viewing the contents of the mesoscale channels in thedevices. The appliance in one embodiment may comprise a microscope forviewing the contents of the mesoscale channels in the devices. Inanother embodiment, a camera may be included in the appliance, asillustrated in the appliance 60 shown schematically in FIGS. 17 and 18.The appliance 60 is provided with a housing 62, a viewing screen 64 anda slot 66 for inserting a device into the appliance. As shown in crosssection in FIG. 18, the appliance 60 also includes a video camera 68, anoptical system 70, and a tilt mechanism 72 for holding device 10, andallowing the placement and angle of device 10 to be adjusted manually.The optical system 70 may include a lens system for magnifying thechannel contents, as well as a light source. The video camera 68 andscreen 64 allow changes in sample fluid properties, such as flowproperties or color, induced by the presence of polynucleotideamplification product, to be monitored visually and optionally recordedusing the appliance. Additionally, addition or removal of fluid samplesto and from the reaction chambers may be monitored, e.g., optically,using the appliance.

In one embodiment, the amplified polynucleotide product can be detectedby using a detection chamber fabricated in the mesoscale flow system inthe substrate in fluid communication with the reaction chamber. Thedetection chamber is provided with a complex-forming agent e.g., abinding moiety capable of binding to the amplified polynucleotide toform a detectable complex. The binding moiety may comprise, e.g., apolynucleotide or antibody probe. The detection chamber may befabricated in accordance with methods disclosed in U.S. Ser. No.07/877,702, filed May 1, 1992, the disclosure of which is incorporatedherein by reference. In another embodiment, the complex-forming agentmay be added to the reaction chamber after the reaction is complete, toform a detectable complex in that chamber. The device may be used incombination with a detector such as an appliance containing amicroprocessor for detecting and recording data obtained during anassay.

In one embodiment, the mesoscale detection chamber may be provided withan inert substrate, e.g., a bead or other particle, capable of bindingto the polynucleotide product, to cause detectable agglomeration of thebeads in the presence of polymerized polynucleotide product. Particleinduced agglomeration can be enhanced by the attachment of a bindingmoiety, such as an antibody, to the particle.

Antibodies or other binding moieties capable of binding to thepolynucleotide product may be introduced into the detection chamber, ormay be coated, either chemically or by adsorption, onto the surface ofthe detection region, or alternatively, onto the surface of an inertparticle in the detection region, to induce binding, giving a positivetest for the polynucleotide. Techniques for the chemical activation ofsilaceous surfaces are well developed, particularly in the context ofchromatography. (See, e.g., Haller in: Solid Phase Biochemistry, W. H.Scouten, Ed., John Wiley, New York, pp 535-597 (1983); and Mandenius etal., Anal. Biochem. 170: 68-72 (1988)). In one embodiment, the bindingmoiety may comprise an antibody, and immunoassay techniques known in theart can be performed in the detection region. (See, e.g., Bolton et al.,Handbook of Experimental Immunology, Weir D. M., Ed., BlackwellScientific Publications, Oxford, 1986, Vol. 1, Chapter 26, for a generaldiscussion of immunoassays).

An optically detectable label such as a fluorescent molecule orfluorescent bead may be attached to the binding moiety to enhancedetection of the amplified polynucleotide product. Alternatively asecond labeled substance, such as a fluorescent labeled antibody may bedelivered through the flow system to bind to the boundpolynucleotide/binding moiety complex in the detection region to producea “sandwich” including an optically detectable moiety indicative of thepresence of the analyte. The binding of the amplified polynucleotide tothe binding moiety in the detection region may be detected, e.g.,optically, either visually or by machine, through a transparent windowdisposed over the detection region. In one embodiment, the production ofamplified polynucleotide may be detected by the addition of a dye suchas ethidium bromide, which exhibits enhanced fluorescence upon bindingto double stranded polynucleotide. Higuchi et al., Biotechnology, 10:413(1992).

The detection chamber may also be provided with a labeled complementarypolynucleotide capable of binding to one of the strands of the amplifiedpolynucleotide, e.g., a labeled polynucleotide immobilized on a bead, toenable the detection of amplified polynucleotide product by means ofbead agglutination. Polynucleotide hybridization techniques known in theart may be utilized. Maniatis et al., Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Press, 1989); Vener et al., Anal.Chem., 198:308-311 (1991). Polynucleotide probes may be attached to,e.g., a submicron latex particle. Wolf et al., Nucleic Acids Research,15:2911-2926 (1987).

In another embodiment, polynucleotide amplification products may beseparated from reactants and other components of the original, sample byelectrophoretic methods adaptable to the mesoscale devices of theinvention. Such techniques are known in the art. For example,microlithographic arrays have been fabricated in SiO₂ for the purpose ofelectrophoretically separating DNA molecules (Volkmuth & Austin, Nature358: 600-602, 1992). Additionally, glass chips have been microfabricatedwith various combinations of channels for performing capillaryelectrophoresis to separate various biological molecules (Harrison etal., Science 261: 895-897, 1993).

In this embodiment, devices of the invention may be fabricated with adetection region comprising a microlithographic array or series ofchannels, and electrophoresis may be performed on the chip by providingan appropriate electric field across the region (e.g., by placingmicroelectrodes at either end of the detection region). The region isprovided at one end with a loading area for collecting the contents ofthe reaction chamber prior to electrophoresis. The various components ofthe reaction mixture are then separated from one another byelectrophoresis. The polynucleotide amplification product may beidentified by size comparison with molecules of known size. In oneembodiment, size markers are introduced to the detection region (by wayof an access port), electrophoretically separated, and the resultsrecorded and stored (e.g. in computer memory). The contents of thereaction chamber are then transferred to the detection region,electrophoretically separated, and the results recorded and comparedwith the results from electrophoresis of the size markers. In thismanner, a polynucleotide amplification product may be identified, aswell as being purified for later use, without the use of inertsubstances and binding moieties for capturing the polynucleotideproduct.

Polynucleotide amplification also can be detected using a detectionregion sensitive to flow restriction caused by the presence ofpolynucleotide produced in the reaction chamber, as is disclosed in U.S.application Ser. No. 08/250,100, the disclosure of which has beenincorporated herein by reference. The presence of amplifiedpolynucleotide also may be detected by sensing the pressure orelectrical conductivity of the fluid samples entering and exiting theflow system. The conductivity may be measured, e.g., using electricalcontacts which extend through the substrate and which mate withelectrical contacts in an appliance used in combination with the device.Electrical contacts can be fabricated by known techniques, such asvarious methods of thermal gradient zone melting. (See Zemel et al., in:Fundamentals and Applications of Chemical Sensors, D. Schuetzle and R.Hammerle, Eds., ACS Symposium Series 309, Washington, D.C., 1986, p. 2.)

Amplified polynucleotide in the reaction chamber can be detected bymonitoring the pressure of the sample fluids. For example, in a device10, nested in appliance 50, illustrated schematically in FIG. 6A, thepressure detectors 54 connected to sample fluid entering and exiting themesoscale flow system through ports 16 will allow the detection ofpressure decreases caused by the presence of polymerized product andresulting clogging or flow restriction. A mesoscale pressure sensor alsomay be fabricated directly on the silicon substrate. Angell et al.,Scientific American 248: 44-55 (1983).

Polynucleotide amplification can be detected by the use of a mesoscaleflow system sensitive to flow restriction, constructed with a “fractal”pattern, i.e., a pattern of diverging flow channels. The channels may befabricated on a silicon substrate to have progressively reduceddimensions, providing progressively narrower flow channels. It will beappreciated by those skilled in the art that, although bifurcatingchannels are exemplified, devices may be fabricated with differentnumbers of parallel flow channels or other symmetrical or asymmetricalpatterns of flow channels with reduced cross-sectional areas.Alternatively, a single channel comprising a narrowed region may beutilized, as described in commonly-owned U.S. application Ser. No.08/250,100 (incorporated by reference herein).

FIG. 7 shows a schematic plan view of a substrate 14 fabricated with asystem of flow channels 40 connected via channel 20 to ports 16 and areaction chamber comprising sections 22A and 22B. The presence ofamplified polynucleotide product in a sample will influence the flowcharacteristics within the flow channels. The channels 40 in thisembodiment are symmetrically disposed and have a progressively narrowerdiameter towards the center of the pattern. Flow through this channelpattern is sensitive to changes in fluid viscosity caused by thepresence of amplified polynucleotide product. Alternatively a morecomplex channel flow system may be utilized, as illustrated in FIG. 13.FIG. 13 illustrates a pair of flow channel systems 40A and 40B. Channelsystem 40A is constructed with progressively narrower flow channelstowards the center of the pattern, resulting in an enhanced sensitivityto flow restriction.

Flow restriction can be detected, e.g., optically, through a transparentcover over the detection region. Alternatively, one or more pressuresensors may be utilized to detect pressure changes due to changes influid properties caused by the accumulation of amplified polynucleotidein or beyond the restricted flow paths. Changes in conductivity uponpolynucleotide amplification also may be readily detected throughelectrical conductivity sensors in contact with the flow region. Forexample, clogging of the restricted region 40, which blocks flow frominlet port 16A to outlet port 16B, could be detected by a conventionalconductivity probe 17 whose output is indicative of the presence orabsence of aqueous fluid in the outflow channel. Binding moieties suchas labeled antibodies or polynucleotide probes may be included in therestricted flow region, e.g., immobilized, or on a solid phase reactantsuch as a bead, to bind to the amplified polynucleotide to induce flowreduction restriction in the restricted flow path.

In one embodiment, the mesoscale flow system includes a chamber forlysing cells from a sample in preparation for downstream polynucleotideanalysis. The devices also may include a region adapted to separate aparticular type of cell in a heterogeneous cell population. The cellseparation region includes binding moieties immobilized on structureswithin the substrate which selectively and reversibly bind a target cellvia a characteristic cell surface molecule such as a protein. Othercells in the sample pass downstream and are channelled into a sump orthrough an exit port. Flow may be continued to wash the cells, e.g.,with a flow of buffer. At higher flow rates and pressures, or bychanging the solvent composition, the washed cells are released from thestructures on which they were immobilized, and thereafter move from thecell separation region downstream to a lysis means, which lyses thecells prior to PCR analysis of intracellular. RNA or DNA.

The cell lysing means typically is disposed in the flow path between thecell separation region (if any) and the polynucleotide amplificationreaction chamber to allow the cells to be lysed prior to analysis for anintracellular polynucleotide. As illustrated in FIG. 9, the cell lysingmeans may comprise cell membrane piercing protrusions 90 extending froma surface of a flow channel 20. As fluid flow is forced through thepiercing protrusion 90, cells are ruptured. In another embodiment, thecell lysis means may simply comprise a region of restrictedcross-sectional dimension which implements cell lysis upon applicationof sufficient flow pressure. The cell lysis means may also comprisesharp edged pieces of silicon trapped within a mesoscale lysis chamber.An appliance which includes means, such as a pump, for forcing the cellcontaining sample into the cell lysis means, causes cell lysis uponapplication of sufficient flow pressure, and subsequently delivers thesample through the flow system to the reaction chamber. In anotherembodiment, the cell lysis means may comprise a cell lysing agent. Celllysing agents known in the art may be utilized.

Reagents may be added to the reaction chamber from a separate inlet portin the substrate in fluid communication with the reaction chamber. Afilter, microfabricated in the flow channel on the substrate, can beused to filter cell debris prior to polynucleotide analysis. In oneembodiment, shown in FIGS. 14, 15 and 16, the filter 24 in device 10 maycomprise a mesoscale flow channel of reduced diameter in comparison withchannel 20. In operation, sample flows from sample flow channel 20Athrough filter 24. Sample filtrate then exits filter 24 and flowsthrough channel 20B. The filter 24 is microfabricated with straight ortortuous channels having preferred depths and widths on the order of 0.1to 50 μm, and span flow channels 20A and 20B, which have maximum depthsand widths on the order of approximately 500 μm. As illustrated in FIG.8, the surface of a flow channel 20 may also include protrusions 80constituting a cellular sieve for separating cells by size upstream fromthe PCR analysis chamber. As cell samples are flowed through the flowchannel, typically under low pressure, only cells small enough to passbetween the protrusions 80 reach downstream functional elements. Thesecells subsequently can be delivered through a cell lysis region, then toa polynucleotide amplification reaction chamber for analysis.

In another embodiment, paramagnetic or ferromagnetic beads may beprovided within the mesoscale flow system, which can be moved along theflow system by an external magnetic field, e.g., in the appliance. Thebeads may be used to transport reagents between functional elements inthe device, or to displace a sample, a reagent or a reaction mixture. Inone embodiment, a polynucleotide probe may be immobilized on themagnetic beads enabling the beads to bind to amplified polynucleotide.Magnetic beads comprising a″ coating of polynucleotide probe may betransported through the flow system to the reaction chamber at the endof an assay to bind to the amplified polynucleotide product. The boundamplified polynucleotide then may be transported on the magnetic beadsto a detection or purification chamber in the flow system, or to acollection port.

G. Exemplary Apparatus

One embodiment of the invention, illustrated in FIG. 10, is a device 10comprising a substrate 14 microfabricated with a mesoscalepolynucleotide amplification chamber comprising sections 22A and 22B,which are connected by flow path 20B. The device 10 is used incombination with an appliance, such as appliance 50, shown in FIG. 6A,which contains a nesting site for holding the device. The appliance 50is provided with flow paths 56 mated to ports 16A, 16B, 16C, and 16D indevice 10. The appliance also includes valves that allow the ports 16A,16B, 16C and 16D to be mechanically opened and closed. Port 16E isincluded for adding reagents to detection chamber 22C. In oneembodiment, the flow systems of the devices may be maintained at ahydraulically full volume, and valves in the appliance, oralternatively, in the devices, may be utilized to direct fluid flow.Sections 22A and 22B of the PCR chamber are heated to, e.g., 94° C. and40-65° C., respectively, to provide a melting temperature and anannealing temperature as required for PCR and other thermally-dependentamplification reactions. As discussed above, reaction chamber sectionsmay be heated by means of an electrical element integrated in thesubstrate below the sections, which can mate with electrical elements inthe appliance. Alternatively, an optical laser may be used to heat thereaction chamber sections through a glass cover disposed over thesubstrate. A heat sensor may be provided in the substrate, in electricalcontact with the appliance. A microprocessor in the appliance can beused to control the temperature of the reaction chamber sections and theflow of fluid in the flow system.

The flow channels of device 10 are fitted with filters 24A, 24B and 24C.Filter 24A is designed to prevent cellular debris and other unwantedparticulate matter in the sample from entering the reaction chambers.Filters 24B and 24C are included for the purpose of restraining thecomplex-forming agent (i.e. beads 92) within detection chamber 22C.Accordingly, filters 24A, 24B and 24C need not be identical.

In operation, for a thermally dependent amplification reaction such asPCR, initially, with the channels and chambers full of buffer, port 16Aand 16C are open while 16B and 16D are closed. A pump 52 in theappliance delivers the sample fluid and/or reagents required foramplification, such as Taq polymerase, primers and nucleosidetriphosphates, via port 16A, through filter 24A, to reaction chambersection 22A. Port 16A next is closed and 16B is opened, and the pump 52in the appliance is used to reciprocate fluid flow in cycles throughflow channel 20B between section 22A, where polynucleotidedehybridization occurs, and section 22B, where annealing andpolymerization occur. Port 16C can be used to vent the system, and alsooptionally to deliver Taq polymerase, nucleoside triphosphates, primers,and other reagents. When the amplification cycling reaction isterminated, e.g., after 30-35 cycles, port 16C is closed, port 16D isopened, and the pump in the appliance is actuated to deliver thereaction products from reaction chamber sections 22A and 22B todetection chamber 22C, which contains, e.g., a polynucleotidecomplementary to the amplified sense and/or antisense strand,immobilized on beads 92. Amplification product is detected by observingthe agglutination of beads 92, e.g., visually through a″ transparentcover disposed over the detection region.

Another embodiment is illustrated in FIG. 10B. The function, structureand operation of this device is similar to that shown in FIG. 10A,except that it comprises a detection region 26, wherein channels orarrays (not shown) may be fabricated for performing electrophoreticseparation of the polynucleotide amplification product. The deviceincludes a port 16E for adding or withdrawing materials from thedetection region. The device is used in combination with an appliancesimilar to appliance 50, shown in FIG. 6A, which further comprises ameans for applying an electric field across detection region 26.

Another embodiment is illustrated in FIG. 11. The function, structure,and operation of this device is identical to that shown in FIG. 10,except that it comprises a single reaction chamber 22A. The device isused in combination with an appliance such as appliance 50 shown in FIG.3A. The device includes means for heating and cooling reaction chamber22A alternatively to a temperature required for melting and atemperature required for annealing and polymerization.

In operation, the appliance is used to deliver a sample containingpolymerase and other reagents required for reactions such as PCR throughinlet port 16A to reaction chamber 22A. Ports 16A and 16D are thenclosed using a valve connected in the appliance. The heating element inthe appliance is then utilized to thermally cycle the reaction chamberbetween a temperature suitable for dehybridization and temperaturessuitable for annealing and polymerization. When the amplification cyclesare terminated, ports 16B and 16D are opened and the sample is deliveredto detection chamber 22B which contains a polynucleotide probe, e.g.,immobilized upon beads 92 or another solid substrate. A positive assayfor the polynucleotide is indicated by agglutination of the solidsubstrate (e.g., beads) in the detection chamber. In the embodimentshown in FIG. 10B, the contents of reaction chamber sections 22A and 22Bare delivered to detection region 26, where the polynucleotide productis electrophoretically separated and identified.

The invention will be understood further from the following, nonlimitingexamples.

Example 1

A polymerase chain reaction is performed in the device illustratedschematically in FIG. 11, provided with a mesoscale reaction chamber22A. To perform a PCR analysis to detect a polynucleotide in a cell, asample cell lysate is added to a buffered solution of Taq polymerase,nucleoside triphosphates, polynucleotide primers and other reagentsrequired for PCR. The cell sample lysate is delivered via the appliancethrough entry port 16A to PCR reaction chamber 22A. Ports 16A and 16Dare closed by means of valves included in the appliance. Amicroprocessor and temperature control element in the appliance are usedto implement a temperature cycle in reaction chamber 22A between 94° C.,for polynucleotide dehybridization, 40-60° C. for annealing and 70-75°C. for primer extension.

After the polymerase chain reaction is complete, ports 16B and 16D areopened, and the pump in the appliance connected to port 168 used todeliver the sample from the PCR reaction chamber 22A through flowchannel 20B to the detection chamber 228. Detection chamber 22B containsbeads 92 comprising a surface immobilized complementary polynucleotidecapable of binding the amplified polynucleotide. The agglutination ofthe beads caused by hybridization reaction between the amplifiedpolynucleotide and the complementary polynucleotide is observed througha window disposed over the detection region 228, and provides a test forthe presence of amplified polynucleotide product.

Example 2

FIG. 12 depicts schematically a device 10 including substrate 14 used toseparate a nucleic acid from a subpopulation of cells in a mixture in abiological fluid sample, and then to perform an assay for a particularnucleotide sequence. Microfabricated on device 10 is a mesoscale flowpath 20 which includes a cell separation chamber 22A, a cell lysischamber 228, filter region 24, a PCR reaction chamber comprisingsections 22C and 22D, and a restricted flow detection region 40. Themesoscale flow system 20 is also provided with fluid entry/exit ports16A, 16B, 16C and 16D. The device is used in combination with anappliance, such as appliance 50, shown in FIG. 6A.

Initially, the valves in the appliance are used to close ports 16C and16D, while ports 16A and 16B are open. A sample containing a mixture ofcells is directed to the sample inlet port 16A by the pump 52 in theappliance, and flows through the mesoscale flow path 20 to separationchamber 22A. Chamber 22A contains binding moieties immobilized on thewall of the chamber which selectively bind to a surface molecule on adesired type of cell in the sample. Remaining cellular components exitthe substrate via port 16B. After binding of the desired cell populationin chamber 22A, flow with buffer is continued, to wash and assureisolation of the cell population. Next part 16B is closed and 16C isopened. Flow is then increased sufficiently to dislodge the immobilizedcells. Flow is continued, forcing cells through membrane piercingprotrusions 90 in chamber 22B, which tear open the cells releasingintracellular material.

Sample flow continues past filter 24, which filters off large cellularmembrane components and other debris, to mesoscale PCR chamber section22C, which is connected to PCR chamber section 22D by flow channel 20B.Taq polymerase, primers and other reagents required for the PCR assaynext are added to section 22D through port 16B from a mated port andflow path in the appliance, permitting mixing of the intracellularsoluble components from the separated subpopulation of cells and the PCRreagents. With port 16A closed, a pump in the appliance connected viaport 16B is used to cycle the PCR sample and reagents through flowchannel 20B between sections 22C and 22D, set at, e.g., 94° C. and 65°C. respectively, to implement plural polynucleotide melting, annealingand polymerization cycles, enabling the amplification of productpolynucleotide. Alternatively, all ports may be closed during theamplification reaction and thermal cycling may be performed as describedin Example 1 above. The valves in the appliance next are used to openport 16D. The pump in the appliance connected to port 16B is then usedto direct the amplified polynucleotide isolated from the cell populationto a detection region comprised of a bifurcating series of flow paths40. Flow restriction in the detection region 40 serves as a positiveindicator of the presence of amplified polynucleotide product and isdetected optically through a glass cover disposed over the detectionregion.

Example 3

The amplification of a sample polynucleotide, (bacteriophage lambda DNA)in a mesoscale reaction chamber, having dimensions of 80 μm in depth, 8mm in width and 14 mm in length, fabricated in a silicon substrate andpassivated using different passivation methods was examined.

To conduct the reaction, PCR reagents (e.g., nucleotides, AmpliTaq DNApolymerase, primer and the bacteriophage lambda DNA sample) were mixedin tubes and transferred to the mesoscale reaction chamber in thesilicon substrate. The final concentrations of the reactants were:nucleotides, 200 mM each, Taq polymerase, 0.25 U/10 ml; primers, 1.0 mMeach; DNA template, 0.1 ng per 10 ml. The thermal cycling (normally 35cycles) was performed automatically using a computer controlled Peltierheater-cooler.

The results of this PCR reaction using different methods of passivationof the walls of the mesoscale reaction chamber fabricated in the siliconsubstrate are illustrated in FIG. 23. FIG. 23 is a drawing of an agarosegel containing ethidium bromide, after electrophoresis of the reactionproducts in the gel. The lanes in the gel correspond as follows: (1 and7) molecular weight markers (1000, 750, 500, 300, 150 and 50 bp); (2)products of a control amplification reaction conducted in a Perkin-ElmerModel 9600 thermal cycler; (3) products of an amplification reaction inan untreated reaction chamber; (4) products of an amplification reactionin the reaction chamber having a thermal silicon oxide film on the wallsurfaces, (5) products of an amplification reaction in the reactionchamber having a silicon nitride coating on the surface formed by aplasma-enhanced chemical vapor deposition (PECVD) process using mixtureof silane and ammonia; and (6) products of an amplification reaction ina reaction chamber having a surface coating of a silicon oxide filmformed by the PECVD process. Methods for the thermal oxidation ofsilicon are described, e.g., in Runyan and Bean, “SemiconductorIntegrated Circuit Processing Technology, “Addison-Wesley PublishingCo., 1990, Chapter 3. Methods for depositing films on surfaces by aplasma-assisted chemical vapor disposition process are described, e.g.,in Sze, “VLSI Technology,” McGraw-Hill Book Co., 1983, Chapter 3.

As illustrated in FIG. 23, the reaction product was substantiallyincreased in the silicon reaction chambers provided with a thermal oxidecoating (lane 4) or a PECVD oxide coating (lane 6) in comparison to theuntreated silicon reaction chamber (lane 3). In contrast, the siliconnitride coating (lane 4) had no positive passivation effect on theamplification reaction.

Example 4

A mesoscale polynucleotide amplification reaction chamber fabricated ina silicon substrate is provided with a coating to passivate the chamberwall surfaces.

A silicon substrate is provided which is fabricated with fluid inlet andoutlet ports and a mesoscale flow system including a flow channel, influid communication with the ports, and a polynucleotide amplificationreaction chamber. The mesoscale amplification reaction chamber, havingdimensions of 80 μm in depth, 8 mm in width, and 14 mm in length, istreated with a siliconizing reagent and optionally a macromolecule toform a coating which passivates the silicon surface. The amplificationchamber is filled with a siliconizing reagent such as AquaSil™ orSurfasil^(ml) (Pierce, Rockford, Ill. or Sigmacote™ (Sigma Chemical Co.,St. Louis, Mo.) using a 100 μl pipette and applying a negative pressureto the exit hole of the chip. The siliconizing reagent is allowed toremain in the chip for at least about 30 min. at room temperature. Aconstant negative pressure is applied to the exit port to remove thesiliconizing reagent, for at least about four hours. About 100 μl ofdistilled water or 0.1 μl TE buffer is delivered through the flow systemvia the inlet port to the amplification chamber, using a 100 μl pipette,and a negative pressure is applied to the exit port. The wash isrepeated about 6 times. After the last wash, negative pressure isapplied to the exit port for about 10 to 15 minutes to drain thechannels.

Alternatively, the amplification chamber surface is passivated with asilanization reagent such as dimethyldichlorosilane (DMDCS) ordimethylchlorosilane (DMCS). Methods which can be used for treatingsurfaces with siliconization or silanization agents are described, e.g.,in Pierce, “Instructions: Siliconizing Agents,” Rockford, Ill., 1993,the disclosure of which is incorporated herein by reference.

The amplification reaction chamber then optionally is filled with asolution of a blocking agent comprising macromolecule (about 10 mg/ml ofmacromolecule in 0.1 M Tris buffer, pH 8.6), e.g., an amino acid polymer(see Table 2), via the inlet port using a 100 μl pipette and applying anegative pressure to the exit port. The macromolecule solution ispermitted to remain in the amplification chamber for at least'about 1 hrat 4° C. A negative pressure then is applied to the exit port of thedevice for about 10 to 15 min. This provides a coating of themacromolecule noncovalently associated with the silicone treatedsurface.

Example 5

The effectiveness of different coatings in diminishing the inhibitoryeffect of silicon on a polynucleotide amplification reaction was tested.

A sample of silicon powder was coated with Surfasil™ (Pierce, Rockford,Ill.) or Sigmacote™ (Sigma Chemical Co., St. Louis, Mo.) and allowed todry. The silicon particles then were coated with a variety of differentmacromolecules (obtained from Sigma Chemical Co., St. Louis, Mo.) listedin Table 2, as described in Example 4. About 4 mg of each coated siliconpreparation was then placed into separate reaction tubes containing 45μl of a PCR reaction mixture (see Example 3) and run in a Perkin ElmerModel 9600 thermal cycler.

Additionally, a mesoscale reaction chamber having dimensions of 80 μm indepth, 8 mm in width, and 14 mm in length, was provided with a coatingof a silanization reagent or siliconization reagent associated withdifferent macromolecules (Table 2), according to the procedure describedin Example 4. A PCR reaction was conducted in the coated reactionchambers using the reagents as described in Example 3. The results usingdifferent coatings are shown in Table 2, using a rating scale of 0 to 4,where the positive control (run in the GeneAmp 9600) has a rating of 3.As illustrated in Table 2, the most effective coating was Surfasil™(Pierce, Rockford, Ill.) in combination with polyvinylpyrrolidone orpolyadenylic acid.

TABLE 2 Rating of Rating of Effectiveness on Effectiveness No. SiliconeAgent/Macromolecule Silicon Powder on PCR Chip 1.Sigmacote ™/Poly-L-alanine 2 - 2. Sigmacote ™/Poly-L-aspartic acid 0 -3. Sigmacote ™/Polyglycine 3 >1  4. Sigmacote ™/Poly-L-leucine 3 0 5.Sigmacote ™/Poly-L-phenylalanine 2 - 6. Sigmacote ™/Poly-L-tryptophan2 - 7. Sigmacote ™/Poly-L-lysine 0 - 8.Sigmacote ™/Polyvinylpyrrolidone >1  - 9. Sigmacote ™/Polyadenylic acid4 0 10. Siginacote ™/Polymaleimide 0 - 11. Sigmacote ™/Maleimide 1 - 12.Surfasil ™/Poly-a-alanine 3 2 13. Surfasil ™/Poly-L-aspartic acid 0 -14. Surfasil ™/Polyglycine 1 - 15. Surfasil ™/Poly-L-leucine 2 - 16.Surfasil ™/Poly-L-phenylalanine 2 - 17. Surfasil ™/Poly-L-tryptophan 1 -18. Surfasil ™/Poly-L-lysine 0 - 19. Surfasil ™/Polyvinylpyrrolidone 4 2to 3 20. Surfasil ™/Polyadenylic acid 4 3 to 4 21.Surfasil ™/Polymaleimide 0 - 22. Surfasil ™/Maleirnide 1 - 23. UncoatedSilicon 0 0 to 2 24. Surfasil ™ 3 0 to 1 25. Sigmacote ™ 2 - 26. DMDCS -0 27. DMDCS/polyadenylic acid - 1 28. AquaSil ™ in H₂O 1:99 - 1

It will be understood that the above descriptions are made by way ofillustration, and that the invention may take other forms within thespirit of the structures and methods described herein. Variations andmodifications will occur to those skilled in the art, and all suchvariations and modifications are considered to be part of the invention,as defined in the claims.

1. A device for amplifying a polynucleotide in a sample by conducting apolynucleotide amplification reaction, the device comprising a solidsubstrate microfabricated to define: a sample inlet port; a mesoscaleflow system comprising; a sample flow channel extending from said inletport; and a polynucleotide amplification region of the device, in fluidcommunication with said flow channel, said region having at least onecross-sectional dimension of width or depth of about 0.1 to 1000microns, at least said polynucleotide amplification region beingpermanently sealed with a cover; and an appliance for use in combinationwith said substrate, said appliance including a nesting site forreceiving said substrate and a conduit in fluid communication with theinlet port of said substrate.
 2. The device of claim 1 wherein thepolynucleotide amplification region comprises a reaction chamber whichhas at least one dissimilar cross-sectional dimension from across-sectional dimension of the sample flow channel.
 3. The device ofclaim 1 wherein said polynucleotide amplification region is configuredto contain at least one reagent for conducting a polymerase chainreaction.
 4. The device of claim 1 wherein said polynucleotideamplification region is configured to contain at least one reagent forconducting a ligase chain reaction.
 5. The device of claim 1 whereinsaid polynucleotide amplification region is configured to contain atleast one reagent for conducting an isothermal DNA amplificationreaction.
 6. The device of claim 1 wherein said polynucleotideamplification region of the device has at least one cross-sectionaldimension of width or depth of about 0.1 to about 500 microns.
 7. Thedevice of claim 1 wherein said polynucleotide amplification region ofthe device has at least one cross:-sectional dimension of width or depthof about 0.1 to about 100 microns.
 8. The device of claim 1 wherein saidpolynucleotide amplification region of the device has at least onecross-sectional dimension of width or depth of about 2 to about 50microns.
 9. The device of claim 1 further including means for thermallycycling the contents of said amplification region whereby in each cyclethe temperature is controlled to dehybridize double strandedpolynucleotide and to permit the in vitro amplification of the samplepolynucleotide.
 10. The device of claim 9, wherein said means forthermally cycling comprises an electromagnetic energy source, saidelectromagnetic energy source being provided in said appliance.
 11. Thedevice of claim 1, further comprising a pump actuable to cause fluidflow through the flow system of said substrate.
 12. A device of claim 1,wherein said pump is disposed in said appliance and connected to saidconduit.
 13. A method for amplifying a polynucleotide in a sample byconducting a polynucleotide amplification reaction, the methodcomprising: (i) providing a device comprising a solid substratemicrofabricated to define: a sample inlet port; a mesoscale flow systemcomprising: a sample flow channel extending from said inlet port; and apolynucleotide amplification region, in fluid communication with saidflow channel, said region having at least one cross sectional dimensionof width or depth of about 0.1 to 1000 microns, at least saidpolynucleotide amplification region being permanently sealed with acover; and an appliance for use in combination with said substrate, saidappliance including a nesting site for receiving said substrate and aconduit in fluid communication with the inlet port of said substrate;(ii) delivering through said conduit, said inlet port and said sampleflow channel to said polynucleotide amplification region, said samplepolynucleotide and at least one reagent required for the polynucleotideamplification reaction; and (iii) thermally controlling the contents ofthe amplification region to amplify said polynucleotide.
 14. The methodof claim 13 wherein said delivering comprises delivering at least onereagent for conducting a polymerase chain reaction.
 15. The method ofclaim 13 wherein said delivering comprises delivering at least onereagent for conducting a ligase chain reaction.
 16. The method of claim13 wherein said delivering comprises delivering at least one reagent forconducting an isothermal DNA amplification reaction.